Effect of Cryopreservation on Olive (Olea europaea L.) Plant Regeneration via Somatic Embryogenesis

Olive somatic embryos have been successfully cryopreserved using the droplet-vitrification method on aluminum foil strips. Although acceptable recovery rates have been obtained after rewarming, the influence of this cryopreservation protocol on the somatic embryogenesis process is unknown. To evaluate the effect of cryopreservation on olive somatic embryogenesis, the behavior of cultures established from cryopreserved somatic embryos was compared with that of control, non-cryopreserved cultures in the different phases of the somatic embryogenesis process. In order to analyze the influence of the genotype, this investigation was carried out in two independent lines. During the proliferation step, only the line T1 was affected by cryopreservation, with higher fresh weight increases. Although similar total embryos were produced per culture, freezing in liquid nitrogen significantly improved the maturation pattern in the line P5. Better germination results were also found in this embryogenic line. The genotype plays a key role, largely determining the effect of cryopreservation on olive somatic embryogenesis. A specific genotype-dependent response was found depending on the culture step. Variations observed could not be associated to differences in the embryogenic lines’ instability to maintain their morphogenic competence after cryopreservation. Embryogenic cultures established after rewarming retained their regeneration capacity, with no evident negative effects affecting their regeneration capacity.

Morpho-histological development of the somatic embryos of Typha domingensis

Background Sustainable methods of propagation of Typha domingensis through somatic embryogenesis can help mitigate its current condition of ecological marginalization and overexploitation. This study examined whether differentiation up to coleoptilar embryos could be obtained in an embryogenic line proliferated with light and high auxin concentration. Methods Murashige and Skoog medium at half ionic strength and containing 3% sucrose and 0.1% ascorbic acid was used for the three embryogenic phases. Induction started with aseptic 9-day-old germinated seeds cultured in 0.5 mg L−1 2,4-dichlorophenoxyacetic (2,4-D). Proliferation of the embryogenic callus was evaluated at 2,4-D concentrations ranging from 0 to 2 mg L−1 in cultures maintained in the dark. The dominant embryogenic products obtained in each treatment were used as embryogenic lines in the third phase. Thus, maturation of the somatic embryos (SEs) was analyzed using four embryogenic lines and under light vs. dark conditions. Embryogenic differentiation was also monitored histologically. Results Proliferation of the nine morphogenetic products was greater in the presence of 2,4-D, regardless of the concentration, than in the absence of auxin. Among the products, a yellow callus was invariably associated with the presence of an oblong SE and suspended cells in the 2,4-D treatments, and a brown callus with scutellar somatic embryos (scSEs) in the treatment without 2,4-D. During the maturation phase, especially the embryogenic line but also the light condition resulted in significant differences, with the highest averages of the nine morphogenetic products obtained under light conditions and the maximum concentration of auxin (YC3 embryogenic line). Only this line achieved scSE growth, under both light and dark conditions. Structurally complete coleoptilar somatic embryos (colSEs) could be anatomically confirmed only during the maturation phase. Discussion In the embryogenic line cultured with the highest auxin concentration, light exposure favored the transdifferentiation from embryogenic callus to scSE or colSE, although growth was asynchronous with respect to the three embryogenic phases. The differentiation and cellular organization of the embryos were compatible with all stages of embryogenic development in other monocotyledons. The growth of colSEs under light conditions in the YC3 embryogenic line and the structurally complete anatomic description of colSEs demonstrated that differentiation up to coleoptilar embryos could be obtained. The diversity of embryogenic products obtained in the YC3 embryogenic line opens up the opportunity to synchronize histological descriptions with the molecules associated with the somatic embryogenesis of Typha spp.

Morphohistological development of the somatic embryo of Typha domingensis

Background. The sustainable methods of propagation for Typha domingensis through somatic embryogenesis can help to mitigate its current condition of ecological marginalisation and overexploitation. Then, the hypothesis established that the variation of the concentration of auxin and light conditions in sequential stages of culture generate different morphogenetic routes that can be monitoring by morphohistological markers. Methods. Murashige and Skoog medium at half ionic strength, 3% sucrose and 0.1% ascorbic acid were used in the induction, proliferation and embryogenic maturation. Induction started with aseptic germinates cultured in 0.5 mg L-1 of 2,4-dichlorophenoxyacetic. Four concentrations of 0 to 2 mg L-1 of 2,4-dichlorophenoxyacetic, that generated four embryogenic lines, were evaluated in darkness. Maturation of the somatic embryo took place, in each embryogenic line, without auxin and under light and dark conditions. Results. The yellow and brown callus, as well as oblong and scutellar somatic embryos were recorded in the methodological sequence. The embryogenic differentiation was described with histological analysis. The induced cultures produced both somatic embryos in a small proportion. The percentages of the yellow callus on the explant and of suspended cells in the embryogenic proliferation were greater with the three concentrations of 2,4-dichlorophenoxyacetic. While, the brown callus predominated without auxin. The somatic embryo developed under light and dark conditions, and presented globular, oblong, scutellar and sparsely coleoptilar stages. Discussion. The combined effect of auxin concentrations and light-dark conditions generated conditions that favoured the development of embryogenic calluses and somatic embryos (globular, oblong, scutellar and coleoptilar) in an asynchronous process with respect to the stages of embryogenic induction, proliferation, and maturation. Indeed, differentiation and cellular organization of this process were compatible with descriptors of the embryogenic stages recorded by other aquatic and terrestrial monocotyledons.

Morphohistological development of the somatic embryo of Typha domingensis

Background. The sustainable methods of propagation for Typha domingensis through somatic embryogenesis can help to mitigate its current condition of ecological marginalisation and overexploitation. Then, the hypothesis established that the variation of the concentration of auxin and light conditions in sequential stages of culture generate different morphogenetic routes that can be monitoring by morphohistological markers. Methods. Murashige and Skoog medium at half ionic strength, 3% sucrose and 0.1% ascorbic acid were used in the induction, proliferation and embryogenic maturation. Induction started with aseptic germinates cultured in 0.5 mg L-1 of 2,4-dichlorophenoxyacetic. Four concentrations of 0 to 2 mg L-1 of 2,4-dichlorophenoxyacetic, that generated four embryogenic lines, were evaluated in darkness. Maturation of the somatic embryo took place, in each embryogenic line, without auxin and under light and dark conditions. Results. The yellow and brown callus, as well as oblong and scutellar somatic embryos were recorded in the methodological sequence. The embryogenic differentiation was described with histological analysis. The induced cultures produced both somatic embryos in a small proportion. The percentages of the yellow callus on the explant and of suspended cells in the embryogenic proliferation were greater with the three concentrations of 2,4-dichlorophenoxyacetic. While, the brown callus predominated without auxin. The somatic embryo developed under light and dark conditions, and presented globular, oblong, scutellar and sparsely coleoptilar stages. Discussion. The combined effect of auxin concentrations and light-dark conditions generated conditions that favoured the development of embryogenic calluses and somatic embryos (globular, oblong, scutellar and coleoptilar) in an asynchronous process with respect to the stages of embryogenic induction, proliferation, and maturation. Indeed, differentiation and cellular organization of this process were compatible with descriptors of the embryogenic stages recorded by other aquatic and terrestrial monocotyledons.

Selection of salt tolerant embryogenic line in Jatropha curcas L., which has potentiality of biodiesel

The embryogenic calli were grown on MS medium containing NaCl with concentrations from 50 to 300 mM. After 2 weeks of culture, salinity tolerance threshold was identified at 150 mM NaCl. Higher concentrations of NaCl stimulated a significant reduction in the calli survival rate and the highest rate was 78.67% at 50 mM. After subculturing callus to the embryo culture medium con- taining NaCl, the growth and embryogenesis were not affected at the concentrations of 50 – 100 mM. Especially, at 50 mM NaCl the embryogenesis rate reached 83.33%. In contrast, 150 mM NaCl inhibited the somatic embryogenesis. After 4 weeks, culturing somatic embryos on medium MS with addition of 0.07 mg/l spermidin at 50 – 100 mM NaCl, the embryogenesis was considered good and embryos developed through several stages: globular, heart, torpedo and cotyledonary. However, at 150 mM NaCl the globular stage appeared in the culture process. The process of morphohistology and using dye carmine – iod and acridine orange observed the structure of generative callus and embryos at several stages. Mô sẹo có khả năng phát sinh phôi được nuôi cấy trong môi trường có chứa muối NaCl với nồng độ thay đổi từ 50 – 300 mM. Sau 2 tuần nuôi cấy, chúng tôi xác định được ngưỡng chịu mặn của mô sẹo có khả năng sinh phôi cây Cọc rào là 150 mM. Nồng độ muối NaCl càng cao thì tỷ lệ sống của mô sẹo giảm dần và đạt giá trị cao nhất là 78,67% tại nồng độ 50 mM NaCl. Khi chuyển mô sẹo sang môi trường phát sinh phôi có chứa muối NaCl với nồng độ thay đổi, chúng tôi thấy ở nồng độ muối NaCl 50 – 100 mM không ảnh hưởng đến khả năng sinh trưởng và phát sinh phôi, đặc biệt là tại nồng độ 50 mM NaCl giúp kích thích sự hình thành phôi từ mô sẹo với tỷ lệ hình thành phôi đạt 83,33%. Ngược lại, nồng độ từ 150 mM NaCl gây ức chế quá trình hình thành phôi soma từ mô sẹo. Tiếp tục khảo sát ảnh hưởng của muối đến khả năng phát triển và nảy mầm của phôi soma. Ghi nhận kết quả sau 4 tuần nuôi cấy phôi soma trong môi trường MS có bổ sung 0.07 mg/l spermidin, tại nồng độ 50 – 100 mM NaCl khả năng hình thành phôi tốt và phôi phát triển qua các giai đoạn phôi hình cầu, hình tim, hình thủy lôi và hình lá mầm. Đặc biệt ở nồng độ 50 mM số lượng phôi lá mầm đạt giá trị cao với 13,33 phôi. Nồng độ muối NaCl 150 mM chỉ xuất hiện phôi hình cầu trong suốt thời gian nuôi cấy. Quá trình giải phẫu hình thái phôi và sử dụng thuốc nhuộm 2 màu carmin – iod và acridine orange đã cho thấy rõ hơn về cấu trúc mô sẹo có khả năng sinh phôi và phôi hình thái.

Analysis of poly(A) + RNA distribution during maize somatic embryogenesis using digoxigenated oligo-dT probes

The pattern of total transcription activity in terms of steady state levels of poly(A)+ containing mRNA during callus initiation and somatic embryogenesis in a high (A188) and a low (A632) embryogenic line of maize was analyzed using digoxigenin labelled oligo-dT probes. A gradual increase and a preferential accumulation of label was observed in both lines, differing temporally up to 4 days in culture. In the A188 line of maize the callus gave rise to somatic embryos. The globular embryos showed less label than the callus; this labelling was mostly present in the basal part of the embryos. At a later stage upper embryogenic and lower non-embryogenic layers were observed in the A188 callus, showing conspicuous differences in the amount of label. In the late globular stage the poly(A)+ RNA signals were seen all over the embryo but at the junction of the suspensor and the callus tissue no label was observed.

StableAgrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

An efficient transformation protocol based on kanamycin selection was developed forAgrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which containedneomycin phosphotransferase II(nptII) as a selectable marker gene andβ-glucuronidase(uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated withAgrobacteriumstrain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for thenptIIanduidAgenes and expression of theuidAgene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.